Project Summary Notch signaling is critical for numerous cell fate decisions in mammals. Mutations that disrupt Notch signaling cause widespread developmental defects and numerous cancers. Therefore, it is critical to understand the factors required for optimal Notch signaling. It is now clear that the glycans added to Notch receptors are key to regulating the level of Notch signaling. Recent experiments identify O-fucose and O-GlcNAc glycans of Notch receptors as those that mediate interactions with canonical Notch ligands, and thereby regulate Notch signal induction. Human diseases and mouse mutants in which O- fucose or O-GlcNAc glycans are reduced reveal that these glycans are not interchangeable. Thus it is important to determine their separate functions. The overall aim of this proposal is to identify specific cell fate decisions regulated by O-fucose versus O-GlcNAc glycans on Notch receptors. Two cell differentiation pathways will be used to address this question - hematopoietic stem cells (HSC) from which all lymphoid and myeloid cell fates develop, and intestinal stem cells (ISC) from which all intestinal crypt-villus cell fates develop. Both HSC and ISC exhibit considerable Notch signaling in the absence of O-fucose glycans. We hypothesize that O-GlcNAc glycans are responsible for this residual Notch signaling via interactions with one or more of the canonical Notch ligands. We will test this hypothesis by determining ligand-induced cell fate decisions following removal or truncation of O-fucose and/or O- GlcNAc glycans in HSC and ISC. Mutant HSC and ISC will be generated by conditionally inactivating genes that encode either the enzyme that adds O-fucose (Pofut1), or one or more Fringe enzymes that extend the fucose with GlcNAc (Lfng, Mfng and Rfng), or the enzyme that adds O-GlcNAc (Eogt). Specific Aim 1 will generate mice with HSC lacking Pofut1 with and without Eogt, or all three Fringe genes with and without Eogt. Lymphoid and myeloid cell fates generated will be analyzed in thymus and spleen. The mechanism of altered differentiation will be investigated by defining the binding of Notch ligands Dll1, Dll4, Jag1 and Jag2 to mutant HSC, the differentiation that each ligand induces in co- culture assays, and the different gene expression consequences. Specific Aim 2 will analyze intestinal cell fates in mice expressing a single Fringe (Lfng, Mfng or Rfng), no Fringe, or all Fringe genes, with and without Eogt. Mice that exhibit altered intestinal cell fate distributions will be investigated by lineage tracing of Lgr5+ cells. Notch ligand binding changes that induce altered cell fate distributions will be identified by binding assays, differentiation in mutant crypt organoids cultured with Dll1, Dll4, Jag1 or Jag2, and gene expression analyses. Interactions of O-GlcNAc glycans with each ligand will be identified in ISC conditionally lacking Pofut1+/-Eogt or all Fringes+/-Eogt by similar in vivo analyses and crypt organoid mechanistic strategies. Important new insights into mechanisms of O-glycan regulation of Notch signaling strength will be obtained.